Study of bioactive secondary metabolites from the marine sponges and marine sponge - associated fungi

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INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR

Chadaporn Prompanya.

Study of Bioactive Secondary Metabolites

from the Marine Sponges and Marine Sponge-Associated Fungi

Study of Bioactive Secondary Metabolites from the Marine Spong

es

and Marine Sponge-Associated Fungi

Chadaporn Prompanya

2018

D

.ICBAS

2018

DOURORAMENTO CIÊNCIAS BIOMÉDICAS

Study of Bioactive

SecondaryMetabolites from the

Marine Sponges and Marine

Sponge-Associated Fungi

Chadaporn Prompanya

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CHADAPORN PROMPANYA

STUDY OF BIOACTIVE SECONDARY METABOLITES FROM THE

MARINE SPONGES AND MARINE SPONGE-ASSOCIATED FUNGI

Thesis submitted to Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto to obtain the degree of Doctor in Biomedical Sciences.

Adviser - Dr. Anake Kijjoa Category - Full Professor

Affiliation - Instituto de Ciências

Biomédicas Abel Salazar da Universidade do Porto

Co-adviser - Dr. Madalena Maria de Magalhães Pinto Category - Full Professor

Affiliation - Faculdade de Farmácia da Universidade do Porto

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The experimental work of this thesis has been carried out in the Departamento de Química, Instituto de Ciências Biomédicas Abel Salazar (ICBAS) da Universidade do Porto. The candidate performed this work with the PhD's scholarship provided by Faculty of Pharmaceutical Sciences of Burapha University, Thailand. This work was partially supported through national funds provided by FCT/MCTES - Foundation for Science and Technology from the Minister of Science, Technology and Higher Education (PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE – Programa Operacional Factores de Competitividade (POFC) programme, under the project PTDC/MAR-BIO/4694/2014 (reference POCI-01-0145-FEDER-016790; Project 3599 – Promover a Produção Científica e Desenvolvimento Tecnológico e a Constituição de Redes Temáticas (3599-PPCDT) in the framework of the programme PT2020 as well as by the project INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, within Research Line NOVELMAR), supported by North Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).

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STATUS THESIS

The results of the work of this thesis have been published, as original articles, in the following journals.

LIST OF PUBLICATIONS

1. Prompanya, C., Dethoup, T., Bessa, L. J., Pinto, M. M., Gales, L., Costa, P. M.,

Silva, A. M., Kijjoa, A. (2014). New isocoumarin derivatives and meroterpenoids from the marine sponge-associated fungus Aspergillus similanensis sp. nov. KUFA 0013. Mar. Drugs, 12, 5160 – 5173. Doi:10.3390/md12105160

2. Prompanya, C., Fernandes, C., Cravo, S., Pinto, M. M., Dethoup, T., Silva, A. M.,

Kijjoa, A. (2015). A new cyclic hexapeptide and a new isocoumarin derivative from the marine sponge-associated fungus Aspergillus similanensis KUFA 0013. Mar.

Drugs, 13, 1432 – 1450. Doi:10.3390/md13031432

3. Prompanya, C., Dethoup, T., Gales, L., Lee, M., Pereira, J. A., Silva, A. M., Pinto,

M. M., Kijjoa, A. (2016). New polyketides and new benzoic acid derivatives from the marine sponge-associated fungus Neosartorya quadricincta KUFA 0081. Mar.

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COMMUNICATIONS

Poster presentations

1. Prompanya, C., Keokitichai, S., Pinto, M., Kijjoa, A. Bromoindoles from Iotrochota

baculifera. Trends in natural products research: young scientists meeting of PSE

and ÖphG. University Centre Obergurgl/Tyrol, Austria, July 21-25, 2013.

2. Prompanya, C., Dethoup, T., Pinto, M., Kijjoa, A. Isocoumarins and cyclic hexapeptide from the sponge-associated fungus Aspergillus similanensis sp. nov. KUFA 0013. 9th_{European Conference on Marine Natural Products. The University}

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INDEX

ACKNOWLEDGEMENTS...i

ABSTRACT...iii

RESUMO...v

LIST OF ABBREVIATIONS AND SYMBOLS...vii

Chapter I. Introduction

1.1) General Introduction...2

1.2) Marine Organisms as a Treasure Source of New Compounds...2

1.3) Marine Sponges are a Potential Source of Novel Compounds...5

1.4) Marine Microbes are the True Treasure Source of Secondary Metabolites...8

1.5) Marine Pharmaceuticals: Approved Drugs and a Current Pipeline Perspective...9

1.5.1) FDA – Approved Drugs...9

1.5.2) Current Marine Pharmaceutical Clinical Pipeline...13

1.6. Aim and Scope of the Study...19

1.6.1) Isolation and Chemical Investigation of Selected Sponge and Fungal Strains...19

1.6.2) Biological Evaluation of the Isolated Compounds...19

Chapter II. Chemistry of the Marine Sponges of the Genus Iotrochota

2.1) The marine sponges of the genus Iotrochota ...21

2.1.1) Iotrochota baculifera...21

2.1.2) Iotrochota birotulata...22

2.1.3) Iotrochota purpurea...24

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INDEX

Chapter III. Chemistry of the Fungi of the Genera Aspergillus and

Neosartorya

3.1) The Fungi of the Genus Aspergillus ... 29

3.1.1) Aspergillus aculeatus...29 3.1.2) Aspergillus clavatus... 30 3.1.3) Aspergillus fumigatus... 31 3.1.4) Aspergillus glaucus...32 3.1.5) Aspergillus insuetus...35 3.1.6) Aspergillus insulicola...35 3.1.7) Aspergillus karnatakaensis...36 3.1.8) Aspergillus niger...37 3.1.9) Aspergillus ochraceus... 40 3.1.10) Aspergillus ostianus...40 3.1.11) Aspergillus parasiticus... 41 3.1.12) Aspergillus sydowii...42 3.1.13) Aspergillus terreus... 43 3.1.14) Aspergillus ustus...47 3.1.15) Aspergillus versicolor...48 3.1.16) Aspergillus wentii...49 3.1.17) Aspergillus westerdijkiae... 51

3.2) The Fungi of the Genus Neosartorya...53

3.2.1) Neosartorya fischeri...53 3.2.2) Neosartorya glabra...62 3.2.3) Neosartorya laciniosa...66 3.2.4) Neosartorya paulistensis...68 3.2.5) Neosartorya pseudofischeri...68 3.2.6) Neosartorya quadricincta...73 3.2.7) Neosartorya siamensis...75 3.2.8) Neosartorya spatulata... 78 3.2.9) Neosartorya spinosa...78 3.2.10) Neosartorya takakii...79

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INDEX

3.2.11) Neosartorya tatenoi... 81

3.2.12) Neosartorya tsunodae... 81

3.2.13) Unspecified Neosartorya species 3.2.13.1) Neosartorya sp...82

3.2.13.2) Neosartorya sp. HN-M-3...83

Chapter IV. Results and Discussion

4.1) The Marine Sponge Iotrochota baculifera...86

4.1.1) 6-Bromo-1H-indole-3-carbaldehyde (40)...86

4.1.2) Methyl (2E)-3-(6-bromo-1H-indol-3-yl)-prop-2-enoate (370)...89

4.2) The Marine-Derived Fungus Aspergillus similanensis KUFA 0013...91

4.2.1) p-Hydroxybenzaldehyde (371)... 91 4.2.2) 6,8-Dihydroxy-3-methylisocoumarin (372)...94 4.2.3) Similanpyrone B (373)...96 4.2.4) Reticulol (374)... 98 4.2.5) Similanpyrone A (375)...101 4.2.6) Similanpyrone C (376)...103 4.2.7) Chevalone C (349)...109 4.2.8) Chevalone E (356)...113 4.2.9) Chevalone B (348)...116 4.2.10) S14-95 (377)...120 4.2.11) Pyripyropene E (154)...124 4.2.12) Pyripyropene S (378)...128 4.2.13) Pyripyropene T (379)... 132 4.2.14) Similanamide (380)...136

4.3) The Marine-Derived Fungus Neosartorya quadricincta KUFA 0081...148

4.3.1) Quadricinctone A (381)...149

4.3.2) Quadricinctapyran A (382)...152

4.3.3) Quadricinctapyran B (383)...155

4.3.4) Quadricinctoxepine (384)... 158

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INDEX 4.3.6) Quadricinctone B (386)...163 4.3.7) Quadricinctone C (387)...167 4.3.8) Quadricinctafuran A (388)...170 4.3.9) Quadricinctafuran B (389)...174 4.3.10) Quadricinctone D (390)...176

4.4) Biological Activity Evaluation of the Isolated Compounds from the Marine Sponge Iotrochota buculifera, and the Marine-Derived Fungi Aspergillus similanensis KUFA 0013 and Neosartorya quadricincta KUFA 0081...184

4.4.1) Antibacterial Activity Evaluation...185

4.4.2) Antifungal Activity Evaluation...187

4.4.3) Cytotoxicity Evaluation... 187

Chapter V. Materials and Methods

5.1) General Experimental Procedures...189

5.2) Isolation and Identification of the Marine-Derived Fungi 5.2.1) Aspergillus similanensis KUFA 0013...190

5.2.2) Neosartorya quadricincta KUFA 0081... 191

5.3) Extraction and Isolation of the Metabolites 5.3.1) The marine sponge Iotrochota baculifera...193

5.3.2) Aspergillus similanensis KUFA 0013...194

5.3.3) Neosartorya quadricincta KUFA 0081...196

5.4) Physical Characteristics and Spectroscopic Data...198

5.5) X-Ray Crystallographic Analysis 5.5.1) X-Ray Crystal Structure of Chevalone E (356)...202

5.5.2) X-Ray Crystal Structure of Quadricinctone A (381), Quadricinctapyran A (382), Quadricinctone B (386), Quadricinctone C (387), Quadricinctafuran A (388) and Quadricinctone D (390)...202

5.6) Molecular Mechanics Conformation Analysis of Quadricinctoxepine (383) and Quadricinctone D (390)...204

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INDEX

5.7) Biological Activity Assays 5.7.1) Cytotoxicity Bioassay

5.7.1.1) Samples...205

5.7.1.2) Cell Cultures...205

5.7.1.3) Cell Growth Inhibitory Assay...205

5.7.2) Antimicrobial Activity Assay 5.7.2.1) Bacterial and Fungal Strains...206

5.7.2.2) Determination of Minimum Inhibitory Bactericidal/Fungal Concentrations...206

5.7.2.3) Synergistic Studies...207

5.7.2.3.1) Screening of Combined Effect the Compounds and Antibiotics...207

5.7.2.3.2) Synergy Test Checkerboard Method...207

5.8) Amino Acids Analysis of Acidic Hydrolysis of Similanamide (380) 5.8.1) Acid Hydrolysis...208

5.8.2) Chiral HPLC Analysis... 208

Chapter VI. Conclusions...210

REFERENCES...215

APPENDICES

Appendix I. NMR Spectra of the Isolated Compounds...236

Appendix II. Prompanya, C., Dethoup, T., Bessa, L. J., Pinto, M. M., Gales, L., Costa, P. M., Silva, A. M., Kijjoa, A. (2014). New isocoumarin derivatives and meroterpenoids from the marine sponge-associated fungus Aspergillus similanensis sp. nov. KUFA 0013. Mar. Drugs, 12, 5160 – 5173. Doi:10.3390/md12105160...263

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INDEX

Appendix III. Prompanya, C., Fernandes, C., Cravo, S., Pinto, M. M.,

Dethoup, T., Silva, A. M., Kijjoa, A. (2015). A new cyclic hexapeptide and a new isocoumarin derivative from the marine sponge-associated fungus Aspergillus similanensis KUFA 0013.

Mar. Drugs, 13, 1432 – 1450. DOI:10.3390/md13031432...278

Appendix IV. Prompanya, C., Dethoup, T., Gales, L., Lee, M.,

Pereira, J. A., Silva, A. M., Pinto, M. M., Kijjoa, A. (2016). New polyketides and new benzoic acid derivatives from the marine sponge-associated fungus Neosartorya quadricincta KUFA

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FIGURES INDEX

Figure 1. Structures of penicillin G (1), paclitaxel (2) and cytarabine (3)...3 Figure 2. Variation in number of new marine natural products for

1985 – 2012...4

Figure 3. The quantity and proportion of bioactive compounds in each

category of chemical compounds...5

Figure 4. Structures of spongothymidine (4), spongouridine (5) and

vidarabine (6)... 6

Figure 5. Structure of trabectedin (7)...9

Figure 6

.

Amino acid sequence of ziconotide

(8),

and structures of

halichondrin A (9), eribulin (10) and brentuximab vedotin (11)...14

Figure 7. Structures of iotroridoside-B (12), sphingolipid (13),

sphingolipid homologues (14a – d), purpurone (15),

baculiferins A-O (16 – 30) and ningalin A (31)...23

Figure 8. Structures of 2β,3β,14α,20β-tetrahydroxy-22α-

(2-hydroxyacetiloxy)-5β-colest-7-en-6-one (32), ponasterone A (33), (2β,3β,5β,22R)-2,3,14,20,24-pentahydroxy-6-oxocholest-7-pen-22-yl-glycolate (34), (2β,3β,5β,22R)2,3,14,20,24-pentahydroxy-6-oxocholest-7-pen-22-yl-acetate (35), ecdysterone (36), 1,3-dibromo-5-(2-[(p-hydroxyphenyl)acetamido]ethyl)-2-[3-(methyl-2-buten-

amido)propoxyl]benzene (37) and β-sitosterol (38)...24

Figure 9. Structures of matemone (39), 6-bromoindole-3-carbaldehyde

(40), itampolins A (41) and B (42), iotrochotamide I (43), iotrochotamide II (44), 6-bromo-1H-indole-3-carboxylic acid methyl ester (45), 6-bromo-1H-indole-3-carboxylic acid ethyl ester (46), purpuroines A – J (47 – 56) and O-methyl-N-trimethyl-3,5-dibromo-

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FIGURES INDEX

Figure 10. Structures of methyl (E)-3-(6-bromoindol-3-yl) prop-2-eoate (58)

and iotrochamides A (59) and B (60)...27

Figure 11. Structures of aspergillusol A (61), 4-hydroxyphenylpyruvic acid

oxime (62), secalonic acid A (63) and asperaculin A (64)...30

Figure 12. Structures of 4,4’ -dimethoxy - 5,5’- dimethyl-7,7’- oxydicoumarin

(65), 7-(γ,γ -dimethylallyloxy) - 5 - methoxy - 4 - methylcoumarin (66), kotanin (67), orlandin (68), (S)-5-hydroxy-2,6-dimethyl-4H-furo[3,4-g]benzopyran-4,8(6H)-dione (69) and 24-hydroxylergosta-

4,6,8(14),22-tetraen-3-one (70)...31

Figure 13. Structures of 9-deacetylfumigaclavine C (71),

9-deacetoxyfumi-gaclavine C (72), fumi9-deacetoxyfumi-gaclavine C (73), 14-norpseurotin (74), pseurotin A (75), spirotryprostatin A (76), 6-methoxyspirotryprostatin B (77), fumitremorgin F (78), dimethylgliotoxin (79), 12α-fumitre-morgin C (80), demethoxyfumitre12α-fumitre-morgin C (81), verruculogen (82)

and tryprostatins A (83) and B (84)...33

Figure 14. Structures of aspergiolide A (85), aspergiolide B (86),

isotora-chrysone (87), isotoraisotora-chrysone-6-O-α-D-ribofuranoside (88), methoxy-3-methyl-1-naphthalenol-6-O-α-D-ribofuranoside (89), 8-methoxy-1-naphthalenol-6-O-α-D-ribofuranoside (90), asperflavin (91), isoasperflavin (92), emodin (93), physcion (94), questin (95), catenarin (96), rubrocristin (97), (+)-variecolor-quinones A (98) physcion bianthrones (99) and (trans)- and (cis)-emodinphyscion

bianthrone (100 and 101)...34

Figure 15. Structures of terretonins E (102) and F (103), and

aurantiamine (104)...35

Figure 16. Structures of insulicolide A (105), asteltoxin (106) and

azonazine (107)...36

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FIGURES INDEX

Figure 18. Structures of cycloleucomelone (110), bicoumanigrin (111),

aspernigrins A (112) and B (113) and pyranonigrins A – D (114 –

117), nigerapyrones A – E (118 – 122), asnipyrones B (123) and A

(124), and nigerapyrones F – H (125 – 127 )...39

Figure 19. Structures of circumdatins A – F (128 – 133)...40

Figure 20. Structures of aspinotriols A (134) and B (135), aspinonediol (136),

aspinonene (137) and dihydroaspyrone (138)...41

Figure 21. Structures of parasitenone (139), 3-chloro-4,5-dihydroxybenzyl

alcohol (140) and gentisyl alcohol (141)...42

Figure 22. Structures of 6-methoxyspirotryprostatin B (142),

18-oxotrypro-statin A (143) and 14-hydroxyterezine D (144), (4S,5S,6S,8S,9S,- 10R,13R,14S,16S,17Z)-6,16-diacetoxy-25-hydroxy-3,7-dioxy-29-nordammara-1,17(20)-dien-21-oic acid (145), terezine D (146), pseustin A (147), helvolic acid (148), 12,13-dihydroxyfumitremorgin C (149), fumitremorgin C (150), didehydrobesdethiobis(methylthio)-gliotoxin (151), fumigaclavine B (152), pyripyropene A (153), pyripyropene E (154), aspergillusenes A (155) and B (156), (+)-(7S)-7-O-methylsydonic acid (157), aspergillusones A (158) and B (159), (+)-(7S)-sydonic acid (160), (7R,8R)-AGI-B4 (161), (7R,8R)-α-diver-sonolic ester (162), methyl 8-hydroxy-6-methyl-9-oxo-9H-xanthene-

1-carboxylate (163), and sydowinins A (164) and B (165)...45

Figure 23. Structures of terremides A (166) and B (167), terrelactone A

(168), 3,4,5-trimethoxy-2-(2-(nicotinamido)benzamido)benzoate (169), (+)-butyrolactones I – III (170 – 172), 3-hydroxy-5-[[4-hydroxy- 3-(3-methyl-2-buten-1-yl)phenyl]methyl]-4-)4-hydroxyphenyl)-2(5H)-furanone (173), aspernolide A (174), 5-[(3,4-dihydro-2,2-dimethyl- 2H-1-benzopyran-6-yl)-methyl]-3-hydroxy-4-(4-hydroxyphenyl)-2(5H)-furanone (175), territrem B (176), (-)-(1R,4R)-1,4-(2,3)-indo- methane-1-methyl-2,4-dihydro-1H-pyrazino[2,1-b]quinazoline-3,6-dione (177), R(-)-6-hydroxymellein (178), trans-4,6-dihydroxymellein

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FIGURES INDEX

Figure 24. Structures of 3β,9α,11-trihydroxy-6-oxodrim-7-ene (183),

2α,9α,11-trihydroxy-6-oxodrim-7-ene (184), 2α,11-dihydroxy-6-oxodrim-7-ene (185), deoxyuvidin B (186), strobilactone B (187), mono(6-strobilactone-B) ester of (E,E)-2,4-hexadienendioic acid (188), (6-strobilactone-B) ester of (E,E)-6-oxo-2,4-hexadienoic acid (189), (6-strobilactone-B) ester of (E,E)-6,7-dihydroxy-2,4-octadie-noic acid (190), (6-strobilactone-B) ester of (E,E)-6,7-dihydroxy-2,4-

octadienoic acid (191) and RES-1149-2 (192)...48

Figure 25. Structures of cottoquinazoline A (193), cotteslosins A (194) and

B (195), sterigmatocystin (196), violaceol I (197), violaceol II (198), diorcinol (199), (-)-cyclopenol (200), viridicatol (201), versicamides A-H (202-209), (-)-enamide (210), notoamide E (211) and

brevianamide F (212)...50

Figure 26. Structures of asperolides A-C (213-215), a tetranorditerpenoid

derivative (216), wentilactones A (217) and B (218), botryosphaerin

B (219) and LL-Z1271-β (220)...51

Figure 27. Structures of circumdatins K (221) and L (222),

5-chlorosclero-tiamide (223), 10-epi-sclero5-chlorosclero-tiamide (224), aspergilliamide B (225), (+)-circumdatin F (226), circumdatin G (227), sclerotiamide (228),

notoamide C (229) and notoamide I (230)...52

Figure 28. Structures of fiscalins A (231), B (232) and C (233)...53 Figure 29. Structures of xanthocillins, NK372135s A-C (234-236),

neosar-torin (237), isoterrein (238), terrein (239), nortryptoquivalone (240)

and aszonalenin (241)...54

Figure 30. Structures of cottoquinazolines E (242) and F (243)...56 Figure 31. Structures of fischeacid (244), fischexanthone (245), AGI-B4

(246), chrysophanol (247), 5’-deoxy-5’-methylamino-adenosine

(248), adenosine (249) and 3,4-dihydroxybenzoic acid (250)...57

Figure 32. Structures of 1-formyl-5-hydroxyaszonalenin (251),

acetylaszonalenin (252), sartorypyrone A (253), sartorypyrone D (254), 13-oxofumitremorgin B (255), aszonapyrone A (256) and

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FIGURES INDEX

aszonapyrone B (257)...59

Figure 33. Structures of 6-hydroxyaszonalenin (258), fumitremorgin B (259), neofipiperazines A – C (260 – 262), verruculogen TR-2 (263), cyclotryprostatin (264), rel-(8S)-19,20-dihydro-9,20-dihydroxy-8-methoxy-9,18-diepifumitremorgin C (265), neofipiperazine D (266), ergosterol (267) and (1S,2R,5R,6R,9R,10R,13S,15S)-5-[2R,3E,5S)-5,6-dimehtyl-3-hepten-2-yl]-6,10-dimethyl-16,17-dioxapentacyclo [12.2.2.01,9_.02,6_.010,15_{]nonadec-18-en-13-ol (268)...61}

Figure 34. Structures of tryptoquivalines T (269) and U (270)...62

Figure 35. Structures of glabramycins A – C (271 – 273)...63

Figure 36. Structures of sartoryglabrins A – C (274 – 276)...64

Figure 37. Structures of neosarphenols A (277) and B (278), methoxyvermi-statin (279), 6-demethylvermimethoxyvermi-statin (280), vermimethoxyvermi-statin (281), penicillide (282), purpactin A (283), phialophoriol (284) and chrodri- manins A (285) and B (286)...65

Figure 38. Structures of (3R)-3-(1H-indol-3-ylmethyl)-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (287), takakiamide (288), (11aR)-2,3- dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (289), sartoryglabramides A (290) and B (291), fellutanine A (292) and fellutamine A epoxide (293)...66

Figure 39. Structures of 3’-(4’oxoquinazoline-3-yl)spiro[1H-indole-3,5’- oxolane]-2,2’-dione (294) and tryptoquivalines L (295) and T (296)...67

Figure 40. Structures of 4(3H)-quinazolinone (297), sartorypyrone C (298), and tryptoquivalines H (299) and F (300)...68

Figure 41. Structures of 3,8-diacetyl-4-(3-methoxy-4,5-methylenedioxy) benzyl-7-phenyl-6-oxa-3,8-diazabicyclo[3.2.1]octane (301a, 301b), pseudofischerine (302), 3-hydroxy-5-methyl-phenyl-2,4-dihydroxy-6-methylbenzoate (303), cadinene (304), eurochevalierine (305), brasiliamide B (306), fischerindoline (307), sequiterpene (308), gliotoxin (309) and bis(dethio)bis(methylthio)gliotoxin (310)...70

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FIGURES INDEX

Figure 42. Stuctures of neosartins A (311) and B (312),

1,2,3,4-tetrahydro-2,3-dimethyl-1,4-dioxopyrazino[1,2-a]indole (313), 1,2,3,4-tetrahy-dro-2-methyl-1,2,3-trioxopyrazino[1,2-a]indole (314), 1,2,3,4-tetra-hydro-2-methyl-1,3,4-trioxopyrazino[1,2-a]indole (315),

N-methyl-1H-indole-2-carboxamide (316), acetylgliotoxin (317), reduced

gliotoxin (318), 6-acetylbis(methylthio) gliotoxin (319), bisdethio-bis(methylthio)gliotoxin (320), didehydrobisdethiobis(methylthio)-

gliotoxin (321), bis-N-norgliovictin (322), and neosartin C (323)...72

Figure 43. Structures of 5-olefin phenylpyropene A (324), phenylpyropenes A (325) and C (326), 13-dehydroxylpyripyropene A (327), 7-deacetyl-pyripyropene A (328), deacetyl-sesquiterpene (329), (1S,2R,4aR,- 5R,8R,8aR)-1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,4a,5,6,7,8,8a-octahydronaphthalene (330), 5-formyl-6-hydro-xy-8-isopro-pyl-2-naphthoic acid (331), 6,8-dihydroxyl-3-((1E,3E)-penta-1,3-dien-1-yl)isochroman-1-one (332), isochaetominine C (333), trichodermamide A (334), indolyl-3-acetic acid methyl ester (335), 1-acetyl-β-carboline (336), 1,2,3,4-tetrahydro-6-hydroxyl-2- methyl-1,3,4-trioxopyra-zina[1,2-a]-indole (337) and fumiquinazoline F (338)...74

Figure 44. Structure of PF1223 (339)...75

Figure 45. Structures of 2,4-dihydroxy-3-methylacetophenone (340), sarto-rymensin (341), tryptoquivaline (342), tryptoquivaline O (343), epi-fiscalin C (344), epi-epi-fiscalin A (345), neoepi-fiscalin A (346), epi- neofiscalin A (347) and chevalones B (348) and C (349)...76

Figure 46. Structures of α-mangostin (350) and mangostin 3-sulfate (351)...78

Figure 47. Structures of 1-hydroxychevalone C (352), 1-acetoxychevalone C (353), 1,11-dihydroxychevalone C (354), 11-hydroxychevalone C (355), chevalone E (356), 2S,4S-spinosate (357), 2S,4R-spinosate (358) and quinadoline A (359)...80

Figure 48. Structures of sartorenol (360) and tryptoquivaline U (361)...80

Figure 49. Structures of tatenoic acid (362)...81

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FIGURES INDEX

Figure 51. Structures of azaspirene (364) and RK-805 (365)...83

Figure 52. Structures of tryptoquivalines P – S (366 – 369)...84

Figure 53. Structures of the isolated metabolites of marine sponge Iotrochota baculifera...86

Figure 54. Structures of the isolated metabolites of marine sponge- associated fungus Aspergillus similanensis KUFA 0013...92

Figure 55. ORTEP diagram of chevalone E (356)...116

Figure 56. Chromatogram of the acidic hydrolysate of similanamide (380)...145

Figure 57. Structures of similanamide (380) and PF1171C...147

Figure 58. Structures of the isolated metabolites of marine sponge- associated fungus Neosartorya quadricincta KUFA 0081...148

Figure 59. ORTEP diagram of quadricinctone A (381)...151

Figure 60. ORTEP diagram of quadricinctapyran A (382)...154

Figure 61. The two minimal energy conformations, C1 and C2, for the structure of quadricinctoxepine (384), with R configuration for C-3...161

Figure 62. ORTEP diagram of quadricinctone B (386)...166

Figure 63. ORTEP diagram of quadricinctone C (387)...170

Figure 64. ORTEP diagram of quadricinctafuran A (388)...173

Figure 65. ORTEP diagram of quadricinctone D (390)...178

Figure 66. Colony on MEA, 7 days, 28 °C obverse (A), reverse (B), asci and ascospores (C) and scanning electron microscope of ascospores (D) of Aspergillus similanensis KUFA 0013...191

Figure 67. Colony on PDA, 7 days, 28 °C obverse (A), asci (B), ascospores (C), aspergilla (D), spores (E) of N. quadricincta KUFA 0081 and Clathria reinwardtii (F)...192

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TABLES INDEX

Table 1. Examples of secondary metabolites derived from sponges with

different bioactivities...6

Table 2. The marine-derived compounds which have been

approved by FDA for treatment human diseases...10

Table 3. Current Marine Pharmaceutical Clinical Pipeline...15

Table 4. 1_{H and}13_{C NMR (DMSO, 300.13 MHz and 75.4 MHz), and}

HMBC assignment for 6-bromo-1H-indole-3-carbaldehyde (40)...88

HMBC assignment for methyl (2E)-3-(6-bromo-1H-indol-3-yl)-

prop-2-enoate (370)...90

Table 6.1_{H and}13_{C NMR )CDCl}

3, 300.13 MHz and 75.4 MHz), and

HMBC assignment for p-hydroxybenzaldehyde (371)...94

Table 7.1_{H and}13_{C NMR (DMSO, 500.13 MHz and 125.8 MHz), and}

HMBC assignment for 6,8-dihydroxy-3-methylisocoumarin )372(...96

Table 8 .1_{H and}13_{C NMR )CDCl}₃_{, 500.13 MHz and 125.8 MHz(, and}

HMBC assignment for similanpyrone B (373)...98

Table 9 .1_{H and}13_{C NMR )CDCl}₃_{, 500.13 MHz and 125.8 MHz), and}

HMBC assignment for reticulol (374)...100

Table 10 .1_{H and}13_{C NMR (CDCl}

3, 500.13 MHz and 125.8 MHz), and

HMBC assignment for similanpyrone A (375(...103

Table 11. 1_{H and}13_{C NMR (CDCl}

3, 500.13 MHz and 125.8 MHz), and

HMBC assignment for similanpyrone C (376)...107

3, 300.13 MHz and 75.4 MHz), and

HMBC assignment for chevalone C (349)...112

3, 500.13 MHz and 125.8 MHz), and

HMBC assignment for chevalone E (356)...115

3, 300.13 MHz and 75.4 MHz), and

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TABLES INDEX Table 15. 1_{H and}13_{C NMR (CDCl} 3, 300.13 MHz and 75.4 MHz), and HMBC assignment for S14-95 (377)...123 Table 16. 1_{H and}13_{C NMR (CDCl} 3, 300.13 MHz and 75.4 MHz), and

HMBC assignment for pyripyropene E (154)...127

Table 17. 1_{H and}13_{C NMR (CDCl}₃_{, 500.13 MHz and 125.8 MHz), and}

HMBC assignment for pyripyropene S (378)...131

HMBC assignment for pyripyropene T (379)...135

HMBC assignment of similanamide (380)...144

Table 20. Chiral HPLC analysis of the acidic hydrolysate of

similanamide (380)...146

3, 300.13 MHz and 75.4 MHz),

HMBC, and NOESY assignments for quadricinctone A (381)...152

Table 22. 1_{H and}13_{C NMR (DMSO, 300.13 MHz and 75.4 MHz),}

HMBC, and NOESY assignments for quadricinctapyran A (382)...155

HMBC, and NOESY assignments for quadricinctapyran B (383)...157

HMBC, and NOESY assignments for quadricinctoxepine (384)...160

Table 25.1_{H and}13_{C NMR (CDCl}

3, 300.13 MHz and 75.4 MHz), and

HMBC assignment for 6-hydroxy-2,2-dimethyl-2,3-dihydro-

4H-chromen-4-one (385)...163

HMBC assignment for quadricinctone B (386)...166

HMBC assignment for quadricinctone C (387)...169

HMBC assignment for quadricinctafuran A (388)...172

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TABLES INDEX

3, 300.13 MHz and 75.4 MHz),

HMBC, and NOESY assignments for quadricinctone D (390)...179

Table 31. Antibacterial efficacy of combined effect of antibiotics

with the compounds (15 μg/disc) against three multidrug-resistant

isolates, using the disc diffusion method...186

Table 32. MIC values of chevalone E (356) in combination with oxacillin

or ampicillin, and the respective FIC index obtained against a MRSA

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SCHEMES INDEX

Scheme 1. Proposed biogenesis of similanpyrone C (376)...108 Scheme 2. Proposed biosynthetic pathways for quadricinctones A (381)

and C (387)...181

Scheme 3. Proposed biosynthetic pathways for quadricinctapyrans A (382)

and B (383), 6-hydroxy-2,2-dimethyl-2,3-dihydro-4H-chromen-4-one (385), quadricinctone B (386), quadricinctafurans A (388), B

(389) and quadricinctone D (390)...182

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i

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my adviser, Professor Dr. Anake Kijjoa who gave me the precious opportunity to pursue my doctoral study in Biomedical Sciences at the Instituto de Ciências Biomédicas Abel Salazar (ICBAS) of the University of Porto, for the continuous support, motivation, recommendation, immense knowledge and for his patience. Without his guidance, I surely could not succeed in doing research and writing this thesis. I am really proud to be a PhD student under his supervision.

Besides my adviser, I would also like to give my sincere appreciation to my co-adviser, Professor Dr. Madalena Maria de Magalhães Pinto of the Faculdade de Farmácia, Universidade do Porto, for her kindness and support.

I am thankful to Dr. Sumaitt Putchakarn, Bangsaen Institute of Marine Science, Burapha University, for collection and identification of the marine sponge, and also to Assistant Professor Dr. Tida Dethoup of the Department of Plant Pathology, Kasetsart University, Bangkok, Thailand, and to Dr. Jamrearn Buaruang of the Division of Environmental Science, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand, for collection of the marine animals, isolation, identification and culture of the marine-derived fungi, as well as for preparation of the extracts for my research work.

I am grateful to Professor Dr. Artur Manuel Soares Silva of the Departamento de Quimica, Universidade de Aveiro, for providing all the 1D and 2D NMR spectra, to Professor Dr. Luís Gales of Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, for the X-ray crystallography analysis, to Dr. Michael Lee of the Department of Chemistry, Leicester University (UK), for providing High Resolution Mass spectra, to Professor Dr. Carla Sofia Garcia Fernandes of Faculdade de Farmácia, Universidade do Porto, for her guidance to make HPLC analyses of amino acids by chiral column and their interpretation, and to Professor Dr. José Augusto Caldeira Pereira of Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, for performing and interpretation of molecular mechanics conformation analysis.

I would like to thank Professor Dr. Paulo Costa of Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, and Dr. Lucinda Bessa of CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, for

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ii

ACKNOWLEDGEMENTS

antibacterial assay, to Professor Dr. Eugénia Pinto, Faculdade de Farmácia, /Universidade do Porto, for antifungal assay, and to Professor Dr. Madalena Pedro, Instituto Superior de Ciências da Saúde do Norte (CESPU), for cytotoxic activity assay.

My sincere thank goes to Mrs. Júlia Bessa, Ms. Sonia Pereira and Mrs. Isabel Silva not only for their technical assistance, but also for mental support and friendship, and to Ms. Sara Cravo for technical support of HPLC analysis of amino acids.

I also thank my lab mates, Dr. Suradet Buttachon, Dr. War War May Zin, Madalena Silva, Decha Kumla, Jidapa Noinart, Renato Pereira, Ângela Ferreira and Tin Shine Aung for helping me in TLC plate preparation, providing an unforgettable friendship and a good atmosphere of working. It was great sharing laboratory with all of you.

I would like to express my sincere appreciation to the former Dean, Associate Professor Dr. Sindhchai Keokitichai, and the current Dean, Associate Professor Dr. Ekarin Saifa, of the Faculty of Pharmaceutical Sciences, Burapha University, for the scholarship, and also to my colleagues and friends in Thailand, especially Associate Professor Dr. Mayuree Tantisira, Dr. Nuttinee Teerakulkittipong, Dr. Anusorn Thampithak, Dr. Naphatson Chanthathamrongsiri, and Dr. Thunchanok Sirirak, for all helps and supports during these years.

Finally, I would like to express my gratitude and love to my beloved parents, Mr. Manit and Mrs. Piyatida Prompanya, and my dear brother, Mr. Thanatad Prompanya, for all the love and supports.

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ABSTRACT

This thesis describes isolation, structure elucidation and biological activity evaluation of secondary metabolites of the marine sponge Iotrochota baculifera, collected from the Gulf of Thailand, as well as those produced by the cultures of the marine-derived fungi, Aspergillus similanensis KUFA 0013, isolated from the marine sponge Rhabdermia sp., and Neosartorya quadricincta KUFA 0081, isolated from the marine sponge Clathria reinwardti. The structures of the isolated compounds were established based on extensive analyses of their 1D and 2D NMR, High Resolution Mass and Infrared spectra. The absolute configurations of the chiral compounds were determined by X-ray crystallographic analysis for chevalone E (348), quadricinctone A (381), quadricinctapyran A (382), quadricinctone B (386), quadricinctone C (387), quadricinctafuran A (388) and quadricinctone D (390). In the case of the cyclohexapeptide, similanamide (380), the configurations of its amino acid constituents were determined by HPLC analysis of its hydrolysate by a Chirobiotic T column using the amino acid standards as controls.

From the ethyl acetate extract of the marine sponge I. baculifera, two previously described bromoindoles, 6-bromo-1H-indole-3-carbaldehyde (40) and methyl (2E)-3-(6-bromo-1H-indol-3-yl)-prop-2-enoate (370), were isolated.

The ethyl acetate extract of the culture of A. similanensis KUFA 0013 furnished three previously undescribed isocoumarin derivatives, similanpyrones A (375), B (373) and C (376), one previously undescribed chevalone derivative, chevalone E (356), two previously undescribed pyripyropene derivatives, pyripyropene S (378) and T (379), and one previously unreported cyclohexapeptide, similanamide (380), together with seven previously reported 6,8-dihydroxy-3-methylisocoumarin (372), reticulol (374), p-hydroxy-benzaldehyde (371), chevalones B (348) and C (349), S14-95 (377) and pyripyropene E (154). Compounds 154, 357, 373 – 376 and 378 – 380 were evaluated for their antimicrobial activity against reference strains of Gram-positive and Gram-negative bacteria and the multidrug-resistant isolates (MRS and VRE) from the environment as well as Candida albicans ATCC 10231. None of the tested compounds exhibited any activity at the highest concentration tested (256 μg/mL). Furthermore, these compounds

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ABSTRACT

were also evaluated for their synergistic capacity with antibiotics against the multidrug-resistant isolates from the environment. However, only chevalone E (356) was found to show synergism with oxacillin against methicillin resistant Staphylococcus aureus (MRSA). In addition, pyripyropene T (379) and similanamide (380) were tested for their

in vitro growth inhibitory activity against MCF-7 (breast adenocarcinoma), NCI-H460

(non-small cell lung cancer) and A373 (melanoma) cancer cell lines. Only similanamide (380) showed weak activity against the three tested cell lines.

From the ethyl acetate extract of the culture of N. quadricincta KUFA 0081, two previously undescribed pentaketides, quadricinctones A (381) and C (387), and seven previously unreported benzoic acid derivatives, quadricinctapyrans A (382) and B (383), quadricinctoxepine (384), quadricinctones B (386) and D (390), and quadricinctofurans A (389) and B (389), were isolated, together with the previously reported 6-hydroxy-2,2-dimethyl-2,3-dihydro-4H-chromen-4-one (385). The isolated compounds were evaluated for their antibacterial activity against reference strains of positive and Gram-negative bacteria and the multidrug-resistant isolates from the environment, and antifungal activity against reference strains of yeast, filamentous fungi and dermatophyte, as well as the in vitro growth inhibitory activity against the MCF-7, NCI-H460 and A375-C5 cancer cell lines by the protein binding dye SRB method, however none of the tested compounds were active in all these assays.

Keywords: Iotrochota baculifera; Aspergillus similanensis KUFA 0013; Neosartorya

quadricincta KUFA 0081; bromoindoles; similanpyrones; isocoumarins; pyripyropenes;

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v

RESUMO

Nesta tese é descrito o isolamento, a elucidação estrutural e a avaliação da atividade biológica de metabolitos secundários da esponja marinha Iotrochota

baculifera, coletada no Golfo da Tailândia, e também produzidos por culturas dos fungos

marinhos Aspergillus similanensis KUFA 0013, isolado da esponja marinha

Rhabdermia sp., e Neosartorya quadricincta KUFA 0081, isolado da esponja marinha Clathria reinwardti. As estruturas dos compostos isolados foram estabelecidas por

análise de espectros de RMN (1D e 2D), massa de alta resolução e espectrometria no infravermelho. A configuração absoluta dos compostos quirais foi determinada por cristalografia de raios X para chevalona E (348), quadricinctona A (381), quadricinctapirano A (382), quadricinctona B (386), quadricinctona C (387), quadricinctafurano A (388) e quadricinctona D (390). No caso do ciclo-hexapeptideo similanamida (380), as configurações dos aminoácidos constituintes foram determinadas por análise cromatográfica (HPLC) do respetivo hidrolisado, usando uma coluna Chirobiotic T e aminoácidos padrão como controlo.

Do extrato de acetato de etilo da esponja marinha I. baculifera, foram isolados dois bromoindolos já descritos, 6-bromo-1H-indole-3-carbaldeído (40) e metil-(2E)-3-(6-bromo-1H-indole-3-il)-prop-2-enoato (370).

O extrato de acetato de etilo da cultura de A. similanensis KUFA 0013 forneceu três derivados isocumarínicos já descritos, similanpironas A (375), B (373) e C (376), um derivado já descrito de chevalona, chevalona E (356), dois derivados não descritos de pyripyropeno, o pyripyropenos S (378) e o T (379), e um derivado ciclo-hexapeptideo não descrito, similanamida (380), juntamente com sete outros compostos já descritos, 6,8-di-hidroxi-3-metillisocumarina (372), reticulol (374), p-hidroxibenzaldeído (371), chevalonas B (348) e C (349), S14-95 (377) e pyripyropeno E (154). Os compostos 154, 357,

373-376 e 378- 380 foram avaliados para atividade antimicrobiana em estipes de referência

de bactérias de Gram-positivo e de Gram-negativo e isolados resistentes a múltiplos fármacos (SRM and ERV) de ambiente, assim como Candida albicans ATCC 10231. Nenhum dos compostos testados exibiu atividade mesmo a alta concentração (256 μg/mL). Estes compostos foram também avaliados para capacidade sinergista com

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RESUMO

antibióticos contra isolados resistentes a múltiplos fármacos de ambiente. Somente chevalona E (356) manifestou sinergismo com oxacilina contra Staphylococcus aureus resistentes à meticilina (SARM).

Adicionalmente pyripyropeno T (379) e similanamida (380) foram testados in vitro para avaliação da atividade inibidora do crescimento das linhas celulares tumorais MCF-7 (adenocarcinoma da mama), NCI-H460 (cancro do pulmão de células não pequenas) e A373 (melanoma).Somente similanamida (380) mostrou fraca atividade contra estas linhas celulares.

Do extrato de acetato de etilo da cultura de N. quadricincta KUFA 0081, foram isolados dois pentacetideos ainda não descritos, quadricinctonas A (381) e C (387), sete derivados do ácido benzóico ainda não descritos, quadricinctapiranos A (382) e B (383), quadricinctoxepina (384), quadricinctonas B (386) e D (390) equadricinctofuranos A (389) and B (389), e a já descrita 6-hidroxi-2,2-dimetil-2,3-di-hidro-4H-chromen-4-ona (385).

Os compostos isolados foram avaliados para atividade antibacteriana em estipes de referência de bactérias de Gram-positivo e de Gram-negativo e isolados resistentes a múltiplos fármacos de ambiente e também atividade antifúngica contra estirpes de referência de leveduras, fungos filamentosos e dermatófitos, assim como para avaliação da atividade inibidora do crescimento da linhas celulares tumorais MCF-7, NCI-H460 e A373, pelo método de SRB. Nenhum dos compostos testados manifestou atividade.

Palavras-chave: Iotrochota baculifera; Aspergillus similanensis KUFA 0013;

Neosartorya quadricincta KUFA 0081; bromoindolos; similanpironas; isocumarínicos;

pyripyropenos; chevalonas; cyclo-hexapeptideo; pentacetideos; derivados do ácido benzóico

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LIST OF ABBREVIATIONS AND SYMBOLS

[3_H]EBOB _[3_{H] Ethynylbicycloorthobenzoate}

[M+H]+ _{Pseudo-molecular ion (Positive ion mode)}

[α]20

D Specific optical rotation at 20 °C for D (sodium) line

® Register or Trademark °C Degree Celsius

13_{C NMR} _{Carbon-13 Nuclear Magnetic Resonance} 1_{H NMR} _{Proton Nuclear Magnetic Resonance}

Å Angstrom

A2780 Human ovarian carcinoma cell line A375-C5 Human melanoma cell line

A549 Human lung carcinoma cell line

Ac Acetyl

ADC Antibody drug conjugate AMP Ampicillin

amu Atomic mass unit

Ara-A Arabino furanosyladenine Ara-C Arabinosyl cytosine

ASCT Autologous stem cell transplant ATCC American Type Culture Collection ATP Adenosine triphosphate

B.C. Before Christ

B16F10 Mouse melanoma cell line BCMA B-cell maturation antigen

BEL-7402 Human hepatocellular carcinoma cell line BGC-823 Human gastric cancer cell line

brd Broad doublet

brs Broad singlet

CAM Chick Chorioallantoic Membrane cAMP Cyclic adenosine monophosphate

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CCDC Cambridge Crystallographic Data Centre CD Cluster of differentiation

CD4 Cluster of differentiation 4 CDK2 Cyclin-dependent kinase-2 CFU Colony-forming unit

CIP Ciprofloxacin

CLSI The Clinical and Laboratory Standards Institute cm Centimeter

c-Met Tyrosine-protein kinase Met CoA Coenzyme A

COSY Correlated spectroscopy

CRPC Castration-resistant prostate cancer CTX Cefotaxime

d Doublet

dd Double doublet

ddd Double double doublet

DEPT Distortionless Enhancement by Polarization Transfer

dL Deciliter

DLBCL Diffuse large B-cell lymphoma DMAPP Dimethylallyl diphosphate DMSO Dimethyl sulfoxide

DMSO-d6 Deuterated dimethyl sulfoxide

DMXBA 3-(2,4-Dimethoxybenzylidene)-Anabaseine DNA Deoxyribonucleic acid

DPPH 2,2-Diphenylpicrylhydrazyl DU145 Human prostate cancer cell line

E Erythromycin

e.g. For example

EC50 Half maximal effective concentration

ECACC European Collection of Authenticated Cell Cultures ED100 One hundred percent effective dose

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ix

ED50 Median effective dose

EGFR Epidermal growth factor receptor

EMBRACE Efficacy and Safety of Belimumab in Black Race Patients with Systemic Lupus Erythematosus

ENPP3 Ectonucleotide pyrophosphatase/phosphodiesterase family member 3

et al. And others ET Ecteinascidin EtOAc Ethyl acetate EtOH Ethanol

FBS Fetal bovine serum

FDA Food and Drug Administration FIC Fractional inhibitory concentration

Fr Fraction

g Gram

GABA Gamma-aminobutyric acid GCC Guanylyl cyclase C

GC-MS Gas chromatography – mass spectrometry GI50 Half maximal growth inhibitory concentration

GluPY Glucose-peptone-yeast extract

GPNMB Glycoprotein nonmetastatic melanoma protein b

h Hour

H1299 Human non-small cell lung cancer cell line hCMEC/D3 Human brain microvascular endothelial cell line HCT-116 Human colon cancer cell line

HCT-8 Human colon cancer cell line

HEK293 Human embryonic kidney cancer cell line HeLa Human cervical cancer cell line

HepG2 Human hepatocellular carcinoma cell line HIV-1 Human immunodeficiency virus type-1 HL-60 Human promyelocytic leukemia cell line

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x

HMBC Heteronuclear Multiple Bond Correlation

hNFAP Heterologous Neosartorya ficheri antimicrobial protein HPLC High-performance liquid chromatography

HRESIMS High-resolution electrospray ionization mass spectrometry Hs683 Human glioblastoma cell line

HSQC Heteronuclear Single Quantum Coherence HuCCA-1 Human lung cholangiocarcinoma cell line HUVEC Human umbilical vein endothelial cell

Hz Hertz

I.D. Inside diameter

IC50 Half maximal inhibitory concentration

IL-2 Interleukin 2 IL-8 Interleukin 8 Inc. Incorporation

IPP Isopentenyl pyrophosphate

IR Infrared

IT Intrathecal

ITS Internal Transcribed Spacer

J Coupling constant in Hz

JNK c-Jun N-terminal protein kinase

K562 Human chronic myeloid leukemia cell line KB Human nasopharyngeal epidermoid cell line

Ki The inhibitor constant

L Liter

L1210 Mouse lymphocytic leukemia cell line L5178Y Mouse lymphoma cell line

LCK Lymphocyte-specific protein tyrosine kinase LC-MS Liquid chromatography – mass spectrometry LIV-1 Zinc transporter SLC39A6

LRRC15 Leucine-rich repeat-containing protein 15 LY6E Lymphocyte antigen 6 complex, Locus E

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xi

m Meter

m Multiplet

m/z Mass per charge

MA Massachusetts

MAGI HeLa CD4+ HIV-1 LTR-β-gal cell

MCF-7 Human breast adenocarcinoma cell line

MD Maryland

MDA-MB-231 Human breast adenocarcinoma cell line Me2CO Acetone

MEA Malt Extract Agar MeOH Methanol

MetAP2 Methionine aminopeptidase-2

mg Milligram

MH Mueller-Hinton agar MHB Mueller-Hinton broth MHz Mega hertz

Mia PaCa-2 Human pancreas cancer cell MIC Minimum inhibitory concentration

min Minute

mL Milliliter

mm Millimeter

MM418c5 Human melanoma cell line MMAE Monomethyl auristatin E MMAF Monomethyl auristatin F mmol Millimole

MO Missouri

MOLT-3 Human acute lymphoblastic leukemia cell line mp Melting point in °C

MRSA Methicillin-resistant Staphylococcus aureus MSS31 Mouse spleen stromal cell

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xii

N Normality

NA Not available

NADH Reduced Nicotinamide adenine dinucleotide

NaPi2b Sodium-dependent phosphate transport protein 2b NCI National Cancer Institute

NCI-H187 Human small cell lung cancer cell line NCI-H460 Human non-small cell lung cancer cell line NFAP Neosartorya fishceri antifungal protein

NFRD NADH-fumarate reductase

ng Nanogram

NJ New Jersey

NK-1 Neurokinin 1

NKT cell Natural killer T cell

nm Nanometer

NMR Nuclear Magnetic Resonance

NOESY Nuclear Overhauser Effect Spectroscopy NSCLC Human non-small cell lung cancer cell line NSCLC-N6L16 Human non-small cell lung cancer cell line OAc Acetoxy

OE21 Human esophageal cancer cell line ORTEP Oak Ridge Thermal Ellipsoid Plot

OX Oxacillin

P388 Murine leukemia cell line

PANC-1 Human pancreas ductal adenocarcinoma cell line PC12 Rat pheochromocytoma cell line

PC-3 Human prostate cancer cell line PDA Potato Dextrose Agar

PLK1 Serine/threonine-protein kinase-1 ppm Part per million

PSMA Prostate-specific membrane antigen

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xiii

RAW264.7 Mouse macrophage cell line RNA Ribonucleic acid

s Singlet

S Streptomycin

SAB Sabouraud dextrose agar SAM S-Adenosyl methionine

SCCHN Squamous cell carcinoma of the head and neck SCLC Small cell lung cancer

SEM Standard error of the mean Sfr Sub-fraction

SGC-7901 Human gastric cancer cell SK-MEL-28 Human melanoma cell line SLITRK6 SLIT and NTRK-like protein 6 SMMC-7721 Human hepatoma cell line

sp. Species

SRB Sulforhodamine B

SW1990 Human pancreatic cancer cell line

t Triplet

T47D Human breast cancer cell line TLC Thin Layer Chromatography TMV Tobacco mosaic virus

U-373 MG Human astrocytoma cell line UK United Kingdom

USA United States of America UV Ultraviolet

UV/VIS Ultraviolet – visible spectroscopy

VA Vancomycin

VEGF Vascular endothelial growth factor VRE Vancomycin-resistant enterococci WE-1990 Human pancreatic cancer cell line WI-38 Normal human lung fibroblast cell

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xiv

XDE Xanthocillin dimethylether

XTE Methoxy-xanthocillin dimethylether δ Chemical shift value in ppm

ΔFIC FIC index

λ Wavelength

μg Microgram

μL Microliter

μM Micromolar

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1

CHAPTER I: INTRODUCTION

CHAPTER I

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1.1) General Introduction

Nature is the source of the pharmaceuticals for treatment many human diseases since ancient time. The discovery of clay tablets revealed that plants have been used as medicines in Mesopotamia since 2400 B.C. Initially, apothecaries have been used medicinal plants in a form of crude extracts, then it was developed to partially purified natural products, and later, to single molecule drugs. Until the late 1980s, the improvement of technologies and techniques moved the focus of drug discovery from nature to chemical synthesis in the laboratory. However, nature is still the valuable source of lead compounds for drug development due to the high diversity of organisms. This is reflected by the number of approved drugs derived from nature. From 1981 to 2010, there were 1,135 approved drugs, and 50 % of these drugs were from natural product origin (David et al., 2015). The examples of well-known drugs which derived from nature are penicillin G (1), an antibiotic isolated from fungus Penicillium notatum; paclitaxel (2), a drug for treatment breast cancer obtained from the bark of Taxus brevifolia; the last example is cytarabine or ara-C (3) (Figure 1), an anticancer drug which was modified from the C-nucleoside isolated from marine sponge Cryptotethya crypta (Jaspars et al., 2016).

1.2) Marine Organisms as a Treasure Source of New Compounds

Not only terrestrial organisms can produce bioactive chemicals that provide the lead compounds in drug discovery process, but marine organisms are also the rich source of bioactive compounds. About 70 % of the earth’s surface is the ocean. The biodiversity of the marine environment is high; thus, the ocean became an interesting source for drug discovery. However, there were only a few studies about marine organisms before 1970 due to low technology in that time. Since the SCUBA diving technologies and deep-sea vehicles (e.g., submarine) were improved, the discovery of marine organisms was raised. Moreover, the laboratory techniques were also developed, leading to the discovery and evaluation of the huge number of marine-derived secondary metabolites (Gerwick & Fenner, 2013; David et al., 2015).

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The secondary metabolites obtained from marine organisms have unique chemical structures and high bioactivities compared to the secondary metabolites derived from terrestrial source (Hu et al., 2015). These are because the extreme conditions under the sea, including temperature, pressure, nutrients, light, salinity and competition (Hasan et al., 2015; Jaspars et al., 2016).

Hundreds of new marine-derived compounds are reported every year. The report from Hu et al. (2015) showed that from 1985 to 2005, the number of new compounds reported annually was less than 600 compounds, but since 2006 the number were increased to more than 800 compounds, and more than 1,000 compounds in 2008 to 2012, as shown in Figure 2. The total number of new marine-derived compounds which were reported from 1985 to 2012 is 16,616 compounds. Among these compounds, 4,196 compounds (25.25 %) are bioactive compounds.

1 3

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Figure 1. Structures of penicillin G (1), paclitaxel (2) and cytarabine (3).

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Figure 2. Variation in number of new marine natural products for 1985 – 2012 (Hu et al.,

2015).

The new compounds were divided into eight categories by chemical structure, including alkaloids, terpenes, ethers and ketals, steroids, lactones, hydroxybenzene and quinones, peptides, and others. As shown in Figure 3, the highest number of new compounds belongs to terpenes, followed by alkaloids, but the highest proportion of bioactive compounds is peptides with 40.85 %, followed by lactones, alkaloids, and hydroxybenzene and quinones with 32.61, 31.49 and 31.36 %, respectively. The major bioactivity of these new compounds is anticancer activity (2,225 compounds; 56 % of the total bioactive compounds), followed by antibacterial activity (521 compounds; 13%) (Hu et al., 2015).

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Figure 3. The quantity and proportion of bioactive compounds in each category of chemical

compounds (Hu et al., 2015).

1.3) Marine Sponges are a Potential Source of Novel Compounds

Marine invertebrates, mainly sponges, tunicates, bryozoans and mollusks provided the majority of marine natural products. Among these organisms, sponges (phylum Porifera) have been recognized as a rich source of novel compounds for drug discovery and development (Laport et al., 2009 and Mehbub et al., 2014; Hu

et al., 2015). In the early 1950s, the nucleosides spongothymidine (4) and

spongouridine (5) were discovered, and the first marine-derived anticancer drug, cytarabine (ara-C; 3), and antiviral drug used against Herpes simplex, vidarabine (ara-A; 6) (Figure 4), were developed from these nucleosides. Since then, the bioactive compounds from sponges became interesting (Laport et al., 2009). Till now, more than 5,300 metabolites have been isolated from marine sponges, and more than 200 compounds are being discovery each year (Agrawal et al., 2016). Metabolites isolated from marine sponges showed many interesting biological activities such as anticancer, antimicrobial and anti-inflammatory activities, as shown in Table 1 (Laport et al., 2009 and Sipkema et al., 2005).

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4 5 6

Figure 4. Structures of spongothymidine (4), spongouridine (5) and vidarabine (6).

Table 1. Examples of secondary metabolites derived from sponges with different

bioactivities.

Compound Compound class Mode of action Species References

Anticancer activity BRS1 Diamino-dihydroxy polyunsaturated lipid Protein kinase C inhibitor Calcareous sponge Sipkema et al, 2005 Adociasulfates Triterpenoid hydroquinones Kinesin motor protein inhibitors Haliclona (aka Adocia) sp.

Discodermolide Linear tetraene

lactone Stabilization of microtubules Discodermia dissolute Spongistatin 1 Bis(spiroacetal) macrolide Tubulin polymerization inhibitor Spongia sp.

Latrunculin A Thiazole macrolide

Actin-depolymerization Latrunculia magnifica Neoamphimedine Pyridoacridine alkaloid Topoisomerase II inhibitor Xestospongia cf carbonaria

Namine D Imidazole alkaloid Nitic oxide

synthetase inhibitor Leucetta cf chagosensis Agelasphin (KRN7000) α-Galactosylceramide NKT cell activator Agelas mauritianus Salicylihalamide A Salicylate macrolide v-ATPase inhibitor Haliclona sp.

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6-Hydroximino-4-en-3-one steroids

Oximated steroid Aromatase

inhibitor Cinachyrella sp. Crambescidins 1-4 Pentacyclic guanidine derivatives Ca2+_channel blocker Crambe crambe Antimicrobial activity Discodermins B, C and D

Cyclic peptides Antibacterial Discodermia

kiiensis

Laport et al., 2009 Acanthosterol

I and J

Sulfated sterols Antifungal Acanthodendrilla

sp. Axinellamines B-D Imidazo-azoloimidazole alkaloids Antibacterial Axinella sp. Spongistatin Polyether macrolide lactone

Antifungal Hyrtios erecta Sipkema et

al., 2005

Antiviral activity

Dragmacidin F Indole alkaloid Antiviral Halicortex sp. Laport et al.,

2009 Papuamides

C and D

Cyclic peptide Antiviral (HIV-1) Theonella

mirabilis Haplosamates A and B Sulfamated steroids Antiviral (HIV-1 integrase inhibitor) Xestospongia sp. Sipkema et al., 2005

Hamigeran B Phenolic macrolide Antiviral (herpes

and polio) Hamigera tarangaensis Laport et al., 2009 2-5A 2’,5’ linked oligonucleotide Interferon mediator

Many sponges Sipkema et

al., 2005 Anti-inflammatory activity Manoalide Cyclohexane sesterterpenoid Phospholipase A2 inhibitor Luffariella variabilis Sipkema et al., 2005

Spongidines A-D Pyridinium

alkaloids Phospholipase A2 inhibitor Spongia sp. Jaspaquinol Diterpene benzenoid Lipoxygenase inhibitor Jaspis splendens

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Subersic acid Diterpene

benzenoid

Lipoxygenase inhibitor

Suberea sp.

Immunosuppressive activity

Simplexides Glycolipids Inhibitor of T-cell

proliferation

Plakortis simplex Sipkema et

al., 2005

Polyoxygenated sterols

Sterols IL-8 inhibitor Dysidea sp.

Contignasterol Oxygenated sterol Histamine

release inhibitor

Petrosia contignata

Pateamine A Thiazole macrolide IL-2 inhibitor Mycale sp.

1.4) Marine Microbes are the True Treasure Source of Secondary

Metabolites

At initial studies of marine organisms, researchers were focusing on macroorganisms such as sponges, tunicates and mollusks. Later, there has been a perception that the isolated secondary metabolites might be produced by the symbiotic or associated microorganisms, and this hypothesis are supported in several cases by experimental and circumstantial evidences.

Zhang et al. (2005) reported marine natural products in clinical trial, which revealed that sponges are an important source of bioactive compounds. However, investigation of the microbes isolated from sponges and other marine invertebrates revealed that they produced the same compounds as their host animals.

Gerwick and Fenner (2013) reported the data of marine-derived agents in the preclinical or clinical trial. At that time, there were nine approved drugs and twelve compounds in clinical trial. These twenty-one agents were isolated from different marine sources, majority from mollusks and sponges. However, these compounds were also reported from symbiotic or associated microorganisms.

In addition, Schofield et al. (2015) have been recently confirmed that ecteinascidin 743 (ET-743) or trabectedin (7) (Figure 5), an anticancer drug which was isolated from the tunicate Ecteinascidia turbinata, was truly produced by

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bacteria Candidatus Endoecteinascidia frumentensis, by sequencing and assembling the genome of this bacterium.

These data support the hypothesis that the original producer of many marine secondary metabolites might be the associated microbes in marine invertebrates because of the same chemicals were found from both marine sources.

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Figure 5. Structure of trabectedin (7).

1.5) Marine Pharmaceuticals: Approved Drugs and a Current

Pipeline Perspective

1.5.1) FDA-approved drugs

To date, there are seven drugs which have been approved by the Food and Drug Administration (FDA) (Table 2). Among them, four drugs are anticancer (Mayer et al., 2010). The indication of the marine-derived drugs, which will be discussed in the next section, reveal that they have potential activity to treat patients in severe state or patients who failed with the prior treatment.

The proportion of approved drugs from marine source is higher than another source. The current rate is seven approved drugs from 28,175 discovered molecular entities, e.g. one drug per 4,025 natural products described. Thus, it is approximately 1.2 to 2.5 – fold higher than the average (one in 5,000 – 10,000 tested compounds) (Jaspars et al., 2016).

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Table 2. The marine-derived compounds which have been approved by FDA for

treatment human diseases (http://marinepharmacology.midwestern.edu/ clinPipeline.htm). Compound name Trademark (approved year) Marine organism origin Chemical class Molecular target Disease Area Cytarabine (Ara-C) Cytosar-U® (1969)

Sponge Nucleoside DNA

polymerase Cancer: Leukemia Vidarabin (Ara-A) Vira-A® (1976)

Sponge Nucleoside Viral DNA

polymerase Antiviral: Herpes simplex virus Omega-3-acid ethyl esters Lovaza® (2004) Fish Omega-3 fatty acids Triglyceride-synthesizing enzymes Hypertriglyceride mia Ziconotide Prialt® (2004)

Cone snail Peptide DNA

polymerase

Pain: severe chronic pain

Trabectedin Yondelis®

(2005)

Tunicate Alkaloid Minor groove

of DNA Cancer: soft tissue sarcoma and ovarian cancer Eribulin mesylate Halaven® (2010)

Sponge Macrolide Microtubules Cancer:

metastatic breast cancer Brentuxima b vedotin Adcetris® (2011) Mollusk/ cyano-bacterium ADC (MMAE) CD30 & microtubules Cancer: anaplastic large T-cell systemic malignant lymphoma and Hodgkin’s disease

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Approved marine-derived drugs

Cytarabine (Ara-C; Cytosar-U®₎

Cytarabine (arabinosyl cytosine; cytosine arabinoside; ara-C; 3) is a synthetic analogue of spongothymidine (4), a nucleoside originally isolated from the Caribbean sponge Cryptotethya crypta (Jaspars et al., 2016; Mayer et al., 2010). The FDA has approved cytarabine to be used with other drugs for treatment of patients with acute lymphoblastic leukemia, acute myeloid leukemia and chronic myelogenous leukemia, and to be used alone to prevent and treat patients with meningeal leukemia (https://www.cancer.gov/about-cancer/treatment/drugs/ cytarabine).

Vidarabine (Ara-A; Vira-A®₎

Vidarabine (6) (arabinofuranosyl adenine or adenine arabinoside, Ara-A) is a synthetic purine nucleoside analogue of spongouridine (5), a nucleoside originally isolated from the Caribbean sponge Tethya crypta. Currently, this compound was obtained from fermentation cultures of the bacterium Streptomyces antibioticus. Vidarabine has been approved by the FDA for treatment of Herpes simplex virus infection (Jaspars et al., 2016; Mayer et al., 2010).

Omega-3-acid ethyl esters (Lovasa®₎

Omega-3-acid ethyl esters are marine products obtained from fish oils, typically from oily fish such as mackerel and anchovy. The FDA approved Lovaza®

on October 11, 2004, as a lipid-regulating agent to reduce triglyceride levels in adult patients with severe (≥500 mg/dL) hypertriglyceridemia (Jaspars et al., 2016; http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/021654s041lbl.pdf).